Retroviral transduction of a mutant methylguanine DNA methyltransferase gene into human CD34 cells confers resistance to O6-benzylguanine plus 1,3-bis(2-chloroethyl)-1-nitrosourea

Abstract

Human CD34 cells express low levels of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) and are sensitive to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Gene transfer of the AGT gene, methylguanine DNA methyltransferase (MGMT), results in only modest BCNU resistance. Recently, an AGT inhibitor, O6-benzylguanine (BG), entered clinical trials. In preclinical studies, BG potentiated the cytotoxic effect of BCNU in tumors but increased toxicity to normal CD34 cells. We transferred a mutant MGMT containing a glycine-to-alanine mutation at position 156, resulting in marked resistance to BG, into Chinese hamster cells; the K562 cell line and human CD34 cells used the retroviral backbone MFG. In each instance, cells expressed increased AGT and were much more resistant to the combination of BG and BCNU than the parental cells or cells transduced with wild-type MGMT. Furthermore, the transduction efficiency in human CD34 cells was in excess of 70%, and the proportion of CD34 transduced cells resistant to the combination was > 30%. Thus, retroviral-mediated transduction of a mutant MGMT into CD34 cells appears to be an effective way to induce selective resistance to a drug combination designed to overcome a significant resistance mechanism to nitrosoureas in tumors.

Figure 1

BG inactivation of wtAGT and ΔAGT. wtMGMT or ΔMGMT transfected CHO (A) or transduced K562 (B) cells were exposed to BG for 1 hr. Cells were harvested immediately, and AGT activity in protein extracts was determined by biochemical assay.

Figure 2

Immunohistochemical detection of AGT in ΔMGMT-transduced K562 cells. K562 cells (1 × 105) were infected with dilutions of amphotropic supernatant collected from Am12ΔMGMT clone 20. At a 1:50 dilution of supernatant, ≈10% of the cells expressed nuclear human AGT when reacted with monoclonal antibody mT3.1.

Figure 3

Clonogenic survival of transduced K562 cells after treatment with BG and/or BCNU. K562 cells transduced with wtMGMT (▪), ΔMGMT (•), and untransduced cells (▾) were treated with 0–40 μM BCNU only; wtMGMT (□) and ΔMGMT (○) were treated with 10 μM BG followed by 0–30 μM BCNU. Cells were plated in methylcellulose in triplicate, and colonies were enumerated in 7 days. Error bars represent mean ± SD.

Figure 4

Western blot analysis of human AGT expression. Protein extracts from CHO cells (lanes 1 and 2) transfected with wtMGMT and ΔMGMT and K562 cells (lanes 3 and 4) and pooled human hematopoietic progenitors (lanes 5 and 6) transduced with wtMGMT, ΔMGMT, or lacZ were separated on a 10% SDS/PAGE gel. Blots were immunoreacted with monoclonal antibody mT3.1, and the 22-kDa human AGT was visualized by chemiluminescent substrate. AGT activity was estimated by correlation of band intensity to a protein extract expressing known levels of AGT (lane 7).

Figure 5

Clonogenic survival of transduced CD34 cells following treatment with BG plus BCNU. CD34 cells were transduced by coculture method (A) or retroviral supernatant in the presence of allogenic stroma (B). Transduced cells were treated with 10 μM BG and 0–40 μM BCNU and plated in methylcellulose in triplicate, and colonies were enumerated in 7–10 days. Data points represent the mean ± SEM of five experiments (A) and three experiments (B) from separate donors. P < 0.001 for the comparison ΔMGMT vs. lacZ.

Figure 6

ΔMGMT integration and expression in human hematopoietic progenitor colonies. (A) Representative PCR amplification of a 152-bp MGMT fragment and a 295-bp human dystrophin fragment as an internal control. Analysis of seven MGMT and one lacZ transduced individual progenitor colonies obtained from methylcellulose on day 10 are shown. An MFG-ΔMGMT plasmid was used as a positive control. (B) Reverse transcription–PCR amplification from pooled methylcellulose colonies as described above. After DNase treatment, a 497-bp retroviral-specific fragment was amplified in the presence of reverse transcriptase (+) but not in the absence of reverse transcriptase (−), indicating complete digestion of DNA. PCR product was detected by hybridization with a ³²P random primed MGMT cDNA.